Biotechnology Overview and DNA Extraction

Biotechnology Overview

• Biotechnology applies molecular methods and living organisms to modify genetic material, creating new substances and functions.
• It's used for medicine, agriculture, species identification, and antibiotic production.

DNA Extraction

• The goal is to isolate DNA from cells, removing proteins, RNA, and other components for high quality, quantity, and purity.
• Techniques include lysis of cells followed by chemical/enzymatic treatments to remove unwanted macromolecules.
• Common techniques are organic extraction (phenol-chloroform), non-organic methods (salting out, proteinase K), and adsorption (silica-gel membrane).

Measuring DNA Quality and Quantity

• Gel electrophoresis: Separates DNA fragments by size for visualization.
• Nanodrop: Measures DNA concentration and purity by absorbance.
• Spectrophotometer: Measures DNA concentration by absorbance at 260 nm.
• Qubit: Detects dsDNA quality using fluorometry.
• The A260/A280 ratio determines DNA quality (DNA absorbs at A260, proteins at A280).

Polymerase Chain Reaction (PCR)

• PCR amplifies specific DNA regions to generate billions of copies in about 2 hours.
• Common applications include gene sequencing, paternity tests, disease diagnosis, and forensic identification.
• It uses a DNA template, primers (forward and reverse), Taq polymerase, nucleotides (dNTPs), and buffer.
• The PCR process occurs in a thermal cycler, with steps of denaturation, annealing, and extension.

Multiplex PCR

• Amplifies multiple DNA targets in a single reaction.
• Useful for simultaneous analysis of several DNA regions.

Nested PCR

• Reduces non-specific binding by performing two PCR reactions.
• Useful for amplifying small or degraded DNA samples, such as forensic samples.

Inverse PCR

• Identifies unknown sequences using known flanking sequences.
• Involves restriction enzyme digestion.
• Used for detecting retroviruses and transposons.

Reverse Transcriptase PCR (RT-PCR)

• Converts mRNA into complementary DNA (cDNA) using reverse transcriptase.
• Used to detect or study gene expression.

Overlap PCR

• Joins multiple DNA fragments by splicing.
• Useful for cloning large DNA fragments or fusing genes.

Amplified Fragment Length Polymorphism (AFLP)

• Selectively amplifies DNA fragments from digested genomic DNA.
• Highly sensitive, used for microsatellite analysis and detecting genetic polymorphisms.

Uses of PCR

• Identifying individuals from small DNA samples.
• Studying ancient DNA from fossils, mummies, or preserved specimens.
• Amplifying DNA for prenatal diagnostics.
• Diagnosing diseases like HIV or COVID-19.
• Basic genetic research.

Gel Electrophoresis (for PCR Analysis)

• Separates DNA fragments by size using an electric current.
• Smaller fragments move faster than larger ones toward the positive electrode.
• Used to visualize and determine the size of DNA fragments.

Primer Design

• Primers are short DNA sequences that help initiate DNA replication.
• Key aspects include length (18-30 nucleotides).

DNA Sequencing

• Determining the order of bases in a DNA strand.
• Has revolutionized the understanding of genetics.
• Applications include human disease studies, genomic studies, and evolutionary studies.

Sanger Sequencing Method

• Known as the "chain termination" method.
• Developed by Frederick Sanger.
• Sequenced DNA by creating complementary DNA strands.

Key Components of Sanger Sequencing

• DNA primer that starts DNA synthesis.
• dNTPs (regular nucleotides: A, T, C, G).
• ddNTPs (special nucleotides that stop DNA synthesis).

Sanger Sequencing STEPS

• PCR amplification.
• DNA fragments generated (each ending with fluorescently tagged ddNTPs).
• Capillary electrophoresis separates the fragments by length.
• Laser detects fluorescent labels, generating a chromatogram to display the DNA sequence.

Human Genome Project

• Large-scale DNA sequencing effort, utilizing Sanger Sequencing.
• Took 13 years to complete.
• Cost $3 billion.

Next Generation Sequencing (NGS)

• Introduced between 2004 and 2006.
• Offers massively parallel sequencing with speed and scalability.
• Useful for entire genomes, targeted regions, RNA, and epigenetic studies.

Advantages of NGS

• Increased speed and accuracy in sequencing.
• Lower cost per megabase compared to Sanger Sequencing.

Applications of NGS

• Rapid whole genome sequencing.
• Deep sequencing of target regions.
• RNA sequencing (RNA-Seq).
• Gene expression analysis and cancer research.
• Identify novel pathogens, genetic variants, and epigenetic factors like DNA methylation.

Sequencing by Hybridization

• Probes and detection of specific DNA sequences
• Commonly for identifying disease related SNPs and chromosomal abnormalities.

Sequencing by Synthesis (SBS)

• Builds on Sanger sequencing, using cyclic processes to incorporate nucleotides into growing DNA chains.

NGS Process

• Involves sample preparation (library creation, DNA fragmentation, adapter ligation).
• Amplification on solid surfaces (beads, silicon) creates DNA clusters.
• Generates data output (millions to billions of short DNA reads for analysis).
• Illumina sequencing (Solexa Technology) is an example of this massively parallel method.

Bioinformatics

• Combines computer science, biology, physics, chemistry, and statistics.
• Primarily to analyze biological datasets (e.g., human genome).
• Undertsand biological processes through computational tools and techniques.

Key Aspects of Bioinformatics

• Big Data Analysis (Large datasets like human genome to identify useful information).
• Transcriptomes (Complete set of RNA molecules).
• Proteomes (Examines the structure and properties of all proteins).

Biological Databases

• Store, organize, and provide access to biological data (e.g., NCBI, GenBank, EMBL-EBI, etc.).
• Important to cross-reference different types of data for easy retrieval.

BLAST Overview

• BLAST (Basic Local Alignment Search Tool) compares biological sequences (DNA, proteins) to find similar sequences in databases.
• Types of BLAST: Blastp (protein-protein), tBlastn (protein-translated nucleotide), Blastn (nucleotide-nucleotide), etc.

BLAST Algorithm

• Heuristic program for rapid, efficient searching.
• Uses shortcuts for fast searching.

Sequence Alignment

• Essential to identify regions of similarity and understand evolutionary relationships.
• Algorithms like Global Alignment (full-length sequences) and Local Alignment (highly similar sections).

Pairwise Alignment

• Compares two sequences.
• Used to identify conserved regions.
• Understand protein/functional relationships.

Multiple Sequence Alignment (MSA)

• Aligns 3 or more sequences.
• Used to find conserved regions.
• Understand evolutionary relationships.

Sequence Homology and Evolution

• Homology (Shared ancestry among sequences).
• Orthologs (Similar genes, same ancestor).
• Paralogs (Within same species, arose due to duplication).

Importance of Alignments

• Comparing sequences.
• Identifying mutations.
• Studying genetic variations related to diseases like cystic fibrosis.